Cysteine proteases associated with human disease states can be grouped into three categories: (1) lysosomal cathepsins; (2) cytosolic calpains and processing enzymes such as interkeukin conveting enzymes; and (3) prokaryotic enzymes with autocatalytic activation. Cathepsins B, H, and L are cysteinyl proteases involved in normal protein degradation. As such, they are generally located in the lysosomes of cells. When these enzymes are found extralysosomaly they have been implicated by use of synthetic substrate technology and by natural endogenous inhibitors as playing a causative role in a number of disease states such as rheumatoid arthritis, osteo arthritis, pneumocystis carinii, schistosomiasis, trypanosoma cruzi, trypanosoma brucei brucei, Crithidia fusiculata, malaria, periodontal disease, tumor metastasis, metachromatic leukodystrophy, muscular dystrophy, etc. For example, a connection between cathepsin B-type enzymes and rheumatoid arthritis has been suggested in van Noorden and Everts, "Selective Inhibition of Cysteine Proteinases by Z-Phe-Ala-CH.sub.2 F Suppresses Digestion of Collagen by Fibroblasts and Osteoclasts," 178 Biochemical and Biophysical Research Communications 178; Rifkin, Vernillo, Kleekner, Auszmann, Rosenberg and Zimmerman, "Cathepsin B and L Activities in Isolated Osteoclasts," 179 Biochemical and Biophysical Research Communications 63; Grinde, "The Thiol Proteinase Inhibitors, Z-Phe-Phe-CHN.sub.2 and Z-Phe-Ala-CHN.sub.2, Inhibit Lysosomal Protein Degradation in Isolated Rat Hepatocytes," 757 Biochimica et Biophysica Acta 15; Mason, Bartholomew and Hardwick, "The Use of Benzyloxycarbonyl[.sup.125 I]iodotyrosylalanyldiazomethane as a Probe for Active Cysteine Proteinases in Human Tissues," 263 Biochem. J. 945; van Noorden, Smith and Rasnick, "Cysteine Proteinase Activity in Arthritic Rat Knee Joints and the Effects of a Selective Systemic Inhibitor, Z-Phe-Ala-CH.sub.2 F," 15 J. Rheumatol. 1525; and van Noorden, Vogels and Smith, "Localization and Cytophotometric Analysis of Cathepsin B Activity in Unfixed and Undecalified Cryostat Sections of Whole Rat Knee Joints," 37 J. Histochemistry and Cytochemistry 617. A connection between cathepsin B and osteo arthritis has been suggested in Delaisse, Eeckhout and Vaes, "In Vivo and In Vitro Evidence for the Involvement of Cysteine Proteinases in Bone Resorption," 125 Biochemical and Biophysical Research Communications 441; a connection between cathepsin B and pneumocystis carinii has been suggested in Hayes, Stubberfield, McBride and Wilson, "Alterations in Cysteine Proteinase Content of Rat Lung Associated with Development of Pneumocystis Carinii Infection," 59 Infection and Immunity 3581; a connection between cysteine proteinases and schistosomiasis has been suggested in Cohen, Gregoret, Amiri, Aldape, Railey and McKerrow, "Arresting Tissue Invasion of a Parasite by Protease Inhibitors Chosen With the Aid of Computer Modeling," 30 Biochemistry 11221. A connection between cysteine proteinases and trypanosoma cruzi, trypanosoma brucei brucei and crithidia fasciculata has been suggested in Ashall, Harris, Roberts, Healy and Shaw, "Substrate Specificity and Inhibitor Sensitivity of a Trypanosomatid Alkaline Peptidase," 1035 Biochimica et Biophysica Acta 293, and/or in Ashall, Angliker and Shaw, "Lysis of Trypanosomes by Peptidyl Fluoromethyl Ketones," 170 Biochemical and Biophysical Research Communications 923. A connection between cysteine proteinases and malaria has been suggested in Rosenthal, Wollish, Palmer and Rasnick, "Antimalarial Effects of Peptide Inhibitors of a Plasmodium Falciparum Cysteine Proteinase," 88 J. Clin. Invest. 1467, and in Rosenthal, Lee and Smith, "Inhibition of a Plasmodium Vinckei Cysteine Proteinase Cures Murine Malaria," 91 J. Clin. Invest. 1052. A connection between cathepsin B and tumor metathesis has been suggested in Smith, Rasnick, Burdick, Cho, Rose and Vahratian, "Visualization of Time-Dependent Inactivation of Human Tumor Cathepsin B Isozymes by a Peptidyl Fluoromethyl Ketone Using a Fluorescent Print Technique," 8 Anti-cancer Research 525. A connection between cathepsin B and cancer has been suggested in Gordon and Mourad, 2 Blood Coagulation and Fibrinolysis 735. A connection between cathepsin B and periodontal disease has been suggested in Cox, Cho, Eley and Smith, "A Simple, Combined Fluorogenic and Chromogenic Method for the Assay of Proteases in Gingival Crevicular Fluid," 25 J. Periodont. Res. 164; Uitto, Larjava, Heino and Sorsa, "A Protease of Bacteroides Gingivalis Degrades Cell Surface and Matrix Glycoproteins of Cultured Gingival Fibroblasts and Induces Secretion of Collagenase and Plasminogen Activator," 57 Infection and Immunity 213; Kunimatsu, Yamamoto, Ichimaru, Kato and Kato, "Cathepsins B, H and L Activities in Gingival Crevicular Fluid From Chronic Adult Periodontitis Patients and Experimental Gingivitis Subjects," 25 J Periodont Res 69; Beighton, Radford and Naylor, "Protease Activity in Gingival Crevicular Fluid From Discrete Periodontal Sites in Humans With Periodontitis or Gingivitis"; 35 Archs oral Biol. 329; Cox and Eley, "Preliminary Studies on Cysteine and Serine Proteinase Activities in Inflamed Human Gingiva Using Different 7-Amino-4-Trifluoromethyl Coumarin Substrates and Protease Inhibitors," 32 Archs oral Biol. 599; and Eisenhauer, Hutchinson, Javed and McDonald, "Identification of a Cathepsin B-Like Protease in the Crevicular Fluid of Gingivitis Patients," 62 J Dent Res 917. A connection between cathepsin B and metachromatic leukodystrophy has been suggested in von Figura, Steckel, Conary, Hasilik and Shaw, "Heterogeneity in Late-Onset Metachromatic Leukodystrophy. Effect of Inhibitors of Cysteine Proteinases," 39 Am J Hum Genet. 371; a connection between cathepsin B and muscular leukodystrophy has been suggested in Valentine, Winand, Pradhan, Moise, de Lahunta, Kornegay and Cooper, "Canine X-Linked Muscular Dystrophy as an Animal Model of Duchenne Muscular Dystrophy: A Review," 42 Am J Hum Genet 352; a connection between cathepsin B and rhinovirus has been suggested in Knott, Orr, Montgomery, Sullivan and Weston, "The Expression and Purification of Human Rhinovirus Protease 3C," 182 Eur. J. Biochem. 547; a connection between cathepsin B and kidney disease has been suggested in Baricos, O'Connor, Cortez, Wu and Shah, "The Cysteine Proteinase Inhibitor, E-64, Reduces Proteinuria in an Experimental Model of Glomerulonephritis," 155 Biochemical and Biophysical Research Communications 1318; and a connection between cathepsin B and multiple sclerosis has been suggested in Dahlman, Rutschmann, Kuehn and Reinauer, "Activation of the Multicatalytic Proteinase from Rat Skeletal Muscle by Fatty Acids or Sodium Dodecyl Sulphate," 228 Biochem. J. 171.
Connections between certain disease states and cathepsins H and C have also been established. For example, cathepsin H has been directly linked to the causative agents of Pneumocystis carinii and in the neuromuscular diseases Duchenne dystrophy, polymyositis, and neurogenic disorders. Stauber, Riggs and Schochet, "Fluorescent Protease Histochemistry in Neuromuscular Disease," Neurology 194 (Suppl. 1) March 1984; Stauber, Schochet, Riggs, Gutmann and Crosby, "Nemaline Rod Myopathy: Evidence for a Protease Deficiency," Neurology 34 (Suppl. 1) March 1984. Similarly, cathepsin C has been directly linked to muscular diseases such as nemaline myopathy, to viral infections, and to processing and activation of bone marrow serine proteases (elastase and granzyme A). McGuire, Lipsky and Thiele, "Generation of Active Myeloid and Lymphod Granule Serine Proteases Requires Processing by the Granule Thiol Protease Dipeptidyl Peptidase I, 268 J. Biol. Chem. 2458-67; L. Polgar, Mechanisms of Protease Action (1989); Brown, McGuire and Thiele, "Dipeptidyl Peptidase I is Enriched in Granules of In Vitro- and In Vivo-Activated Cytotoxic T Lymphocytes," 150 Immunology 4733-42. The Brown et al. study effectively demonstrated the feasibility of inhibiting cathepsin C (DPP-I) in the presence of other cysteinyl enzymes based on substrate specificity. Unfortunately, the diazoketones used in that study are believed to be mutagenic and not appropriate for in vivo application.
The cytosolic or membrane-bound cysteine proteases called calpains have also been implicated in a number of disease states. For example, calpain inhibitor can be useful for the treatment of muscular disease such as muscular dystrophy, amyotrophy or the like, 25 Taisha (Metabolism) 183 (1988); 10 J. Pharm. Dynamics 678 (1987); for the treatment of ischemic diseases such as cardiac infarction, stroke and the like, 312 New Eng. J. Med. 159 (1985); 43 Salshin Igaku 783 (1988); 36 Arzneimittel Forschung/Drug Research 190, 671 (1986); 526 Brain Research 177 (1990); for improving the consciousness disturbance or motor disturbance caused by brain trauma, 16 Neurochemical Research 483 (1991); 65 J. Neurosurgery 92 (1986); for the treatment of diseases caused by the demyelination of neurocytes such as multiple sclerosis, peripheral nervous neuropathy and the like, 47 J. Neurochemistry 1007 (1986); and for the treatment of cataracts, 28 Investigative Ophthalmology & Visual Science 1702 (1987); 34 Experimental Eye Research 413 (1982); 6 Lens and Eye Toxicity Research 725 (1989): 32 Investigative Ophthalmology & Visual Science 533 (1991).
Calpain inhibitors may also be used as therapeutic agents for fulminant hepatitis, as inhibitors against aggregation of platelet caused by thrombin, 57 Thrombosis Research 847 (1990); and as a therapeutic agent for diseases such as breast carcinoma, prostatic carcinoma or prostatomegaly, which are suspected of being caused by an abnormal activation of the sex hormone receptors.
Certain protease inhibitors have also been associated with Alzheimer's disease. See, e.g., 11 Scientific American 40 (1991). Further, thiol protease inhibitors are believed to be useful as anti-inflammatory drugs, 263 J. Biological Chem. 1915 (1988); 98 J. Biochem. 87 (1985); as antiallergic drugs, 42 J. Antibiotics 1362 (1989); and to prevent the metastasis of cancer, 57 Seikagaku 1202 (1985); Tumor Progression and Markers 47 (1982); and 256 J. Biological Chemistry 8536 (1984).
Further, Interleukin 1-.beta.-Converting Enzyme (ICE) has been shown to be a cysteine protease implicated in the formation of the cytokine IL-1.beta. which is a potent mediator in the pathogenesis of chronic and acute inflammatory diseases. Tocci and Schmidt, ICOP Newsletter, September 1994. Inhibitors to this enzyme have recently been reported, including Thornberry, Peterson, Zhao, Howard, Griffin, and Chapman, Inactivation of Interleukin-1.beta.-Converting Enzyme by Peptide (Acyloxy)methyl Ketones, 33 Biochemistry 3934 (1994); Dolle, Singh, Rinker, Hoyer, Prasad, Graybill, Salvino, Helaszek, Miller and Ator, "Aspartyl .alpha.-((1-Phenyl-3-(trifluoromethyl)-pyrazol-5-yl)oxy)methyl Ketones as Interleukin-1.beta. Converting Enzyme Inhibitors: Significance of the P.sub.1 and P.sub.3 Amido Nitrogens for Enzyme-Peptide Inhibitor Binding" 37 J. Med. Chem. 3863; Mjalli, Chapman, MacCoss, Thornberry, Peterson, Activated Ketones as Potent Reversible Inhibitors of Interleukin-1.beta.-Converting Enzyme" 4 Biooganic & Medicinal Chemistry Letters, 1965; and Dolle, Singh, Whipple, Osifo, Speier, Graybill, Gregory, Harris, Helaszek, Miller and Ator "Aspartyl .alpha.-((Diphenylphosphinyl)-oxy)-methyl Ketones as Novel Inhibitors of Interleukin-1.beta.-Converting Enzyme: Utility of the Diphenylphosphionic Acid Leaving Group for the Inhibition of Cysteine Proteases" 38 J. Med Chem. 220.
The most promising type of cysteine proteinase inhibitors have an activated carbonyl with a suitable .alpha.-leaving group fused to a programmed peptide sequence that specifically directs the inhibitor to the active site of the targeted enzyme. Once inside the active site, the inhibitor carbonyl is attacked by a cysteine thiolate anion to give the resulting hemiacetal, which collapses via a 1,2-thermal migration of the thiolate and subsequent displacement of the .alpha.-keto-leaving group. The bond between enzyme and inhibitor is then permanent and the enzyme is irreversibly inactivated.
The usefulness of an inhibitor in inactivating a particular enzyme therefore depends not only on the "lock and key" fit of the peptide portion, but also on the reactivity of the bond holding the .alpha.-leaving group to the rest of the inhibitor. It is important that the leaving group be reactive only to the intramolecular displacement via a 1,2-migration of sulfur in the breakdown of the hemithioacetal intermediate.
Groundbreaking work regarding cysteine proteinase inhibitors having an activated carbonyl, a suitable .alpha.-leaving group and a peptide sequence that effectively and specifically directs the inhibitor to the active site of the targeted enzyme was disclosed in U.S. Pat. No. 4,518,528 to Rasnick, incorporated herein by reference. That patent established peptidyl fluoromethyl ketones to be unprecedented inhibitors of cysteine proteinase in selectivity and effectiveness. The fluoromethyl ketones described and synthesized by Rasnick included those of the formula: ##STR1## wherein R.sub.1 and R.sub.2 are independently selected from the group hydrogen, alkyl of 1-6 carbons, substituted alkyl of 1-6 carbons, aryl, and alkylaryl where the alkyl group is of 1-4 carbons; n is an integer from 1-4 inclusive; X is a peptide end-blocking group; and Y is an amino acid or peptide chain of from 1-6 amino acids.
Peptidylketone inhibitors using a phenol leaving group are similar to the peptidyl fluoroketones. As is known in the art, oxygen most closely approaches fluorine in size and electronegativity. Further, when oxygen is bonded to an aromatic ring these values of electronegativity become even closer due to the electron withdrawing effect of the sp.sup.2 carbons. The inductive effect of an .alpha.-ketophenol versus an .alpha.-ketofluoride when measured by the pKa of the .alpha.-hydrogen, appears comparable within experimental error.
Unfortunately, the leaving groups of prior art inhibitors that use a phenoxy group present problems of toxicity, solubility, etc. Solubility is of particular importance in the field of peptide derived drugs where bioavailability becomes the major criterion for the success of a drug. The solubility recommendation of the FDA is 5 mg/mL. Successful in vivo utility of prior art inhibitors has been limited due to the insolubility of the leaving groups. In vivo application to date has centered on inhibitors with peptide requirements allowing ester, acid or free amine side chains as those required in the inhibition of Interleukin-1.beta.-converting enzyme: Revesz, Briswalter, Heng, Leutwiler, Mueller and Wuethrich, "35 Tetrahedron Letters 9693.
International application WO 93/09135 disclosed inhibitors again designed for Interleukin-1.beta.-converting enzyme where an N-hydroxytetrazole was disclosed as a leaving group. Further, tetrazoles have also been used in other pharmaceutical products such as Ceforanide, etc.
The in vivo inhibition of other cysteine proteases using oxygen anionic leaving groups was first disclosed by Zimmerman, Bissell, and Smith in U.S. Pat. No. 5,374,623 where it was disclosed that bioavailability is enhanced by the use of peptidyl .alpha.-aromatic ether methyl ketones with selective peptide combinations not requiring the presence of a free amine or acid side chain. Later, a peptidyl (acyloxy)methyl ketone with lysine in the side chain was reported to have in vivo efficacy: Wagner, Smith, Coles, Copp, Ernest and Krantz, "In Vivo Inhibition of Cathepsin B by Peptidyl (Acyloxy)methyl Ketones," 37 J. Med. Chem. 1833. Unfortunately, peptidyl (acyloxy)methyl ketones are esters that are also subject to cleavage by esterases which makes the .alpha.-ketoethers the preferred construction for cysteine protease inhibitors.
It can be seen from the foregoing that a need continues to exist for cysteine protease inhibitors with improved solubility and toxicity profiles, and which are particularly suitable for in vivo use. The present invention addresses that need.